The pharmacology and molecular pharmacology of various gastrointestinal (GI) peptides are being investigated. Two peptide receptor families investigated during the year are those for bombesin- (Bn) related peptides and for VIP-related peptides. Bn-related peptides (GRP, NMB) interact primarily with two distinct receptors (GRP-R, NMB-R) to mediate a number of effects in the GI tract and central nervous sytem (CNS). The aim of one aspect of this project is to understand the pharmacology, molecular pharmacology, and cell biology of these receptors. Recently, studies in other G protein-coupled receptors (GPC-R) show the 2nd, as well as the 3rd, intracellular loop (IC loop) domains are important for receptor activation and receptor-mediated modulatory processes. Using site-directed mutagenesis we examined the importance of the 2nd IC loop of the GRP-R for receptor affinity, activation, and receptor modulatory processes (internalization, down-regulation). Each residue from 142 to 148 in the GRP-R was altered alone or in combination. Our results show the amino acids have a minimal effect on receptor affinity, but especially IM-143,147AA double mutant had decreased internalization, three mutants showed decreased receptor down-regulation and five had decreased ability to activate phospholipase C (PLC). Correlations between these processes show that GRP-R internalization and chronic down-regulation have a different dependence on PLC activation and are largely independent processes. A second aspect of our studies is to develop receptor antagonists or cytotoxic agents coupled to ligands for Bn or VIP receptors that could have anti-tumor activity. Many tumors overexpressed human Bn receptors (colon, prostate, breast, lung, gastric, ovarian). We attempted to develop a high affinity ligand that interacted with all Bn receptor subtypes coupled to camptothecin (CPT) that could be used as a prodrug for site-specific cytoxicity. One analogue, CPT-L2-BA3, was identified which was a full agonist for all three Bn receptor subtypes, was rapidly internalized, and showed receptor-specific cytoxicity for NCI 1299 lung cancer cells overexpressing these receptors. This analogue should be a useful prototype to explore the effectiveness of tumor-specific cytoxicity delivery using a Bn receptor-mediated mechanism.